The aim of the proposed research is to study the pathways and mechanisms of receptor-mediated endocytosis. Three ligand-receptor systems present in rat hepatocytes but having different fates, were chosen for comparative study: the asialo-glycoprotein, epidermal growth factor, and dimeric-immunoglobulin A systems. 125I-ligands will be injected in vivo and the rates of clearance from circulation, appearance and metabolism in liver will be measured. Then, the intracellular pathway of each ligand will be followed in vivo and in isolated, perfused livers, using 125I-ligands, electron microscope (EM) autoradiography and higher resolution EM tracers (i.e., ligands chemically conjugated to cytochemical and electron dense molecules). Combinations of ligands will be followed by EM analysis in order to, 1) determine the extent of overlap in the pathways of ligands having similar or different intracellular fates, and 2) identify possible sites in which ligands and receptors dissociate (if they do). Next, the effect of temperature and selected inhibitors on each ligand's pathway will be studied. Three temperature ranges to be studied: 4-10 C (internalization blocked); 11-15 C (?); 16-20 C (entry of asialoglycoproteins into lysosomes blocked). The inhibitors are: N-ethyl-maleimide and phenylglyoxal (reported to block uptake); transglutaminase inhibitors (reported to block receptor "clustering"); and "lysosomotropic" agents (reported to block receptor recycling). Specific EM tracers will be used to assess the surface distribution, redistribution, and entry sites (coated pits?) of the bound ligands after exposure of livers to conditions blocking uptake. Common and distinct mechanisms regulating the three ligand receptors will be established, and compartments found to accumulate ligand isolated and biochemically characterized. Finally, the three membrane receptors will be purified, specific antibodies prepared, and kinetic experiments conducted to trace the intracellular fates and pathways of the surface receptors after interaction with ligand. Exogenous labeling reagents will be used (125I-lactoperoxidase) to "mark" surface receptors, followed by subcellular fractionation at various times and analysis of the rates at which labeled receptors (and ligands) are found in different intracellular compartments.